GLUT4 translocation by insulin in intact muscle cells: detection by a fast and quantitative assay

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 mu M) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation, We further show that wortmannin and the cytoskeleton disrupters cytochalasin D and latrunculin B completely blocked these insulin effects, The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs. (C) 1998 Federation of European Biochemical Societies.